Link to my first crappy paper

http://www.jstage.jst.go.jp/article/dsj/6/0/6_S743/_article

Experiment terminologies

1. Macroevolutionary test: the test across species
2. positional cloning methods.

Molecular technologies

1. To prove something is transcription factor: fuse it with a promoter, and look at the downstream luciferase activity
2. interaction between different proteins: Protein precipitation

Roderick MacKinnon

The Nobel Prize in Chemistry 2003
http://nobelprize.org/nobel_prizes/chemistry/laureates/2003/mackinnon-autobio.html

Potassium channel -Kv2.1

http://www.sciencedirect.com.ezproxy.hsclib.sunysb.edu/science?_ob=ArticleURL&_udi=B6W81-4GC1R5J-3&_user=334567&_coverDate=10%2F31%2F2005&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000017318&_version=1&_urlVersion=0&_userid=334567&md5=5c696ee8356d440545693b7d60914a00
The function of Kv channels can be described in simple terms using the biophysical properties that determine
1. at which membrane potentials the channels will open,
2. how quickly they open in response to the membrane potential achieving these potentials,
3. if open how long they remain so, and
4. when open at what rate do they allow flux of K+ across the membrane (MacKinnon, 2003). While these inherent biophysical properties are clearly encoded within the primary structure of the particular channel subtype, they can also be modified through post-translational events including covalent modifications (usually phosphorylation) and non-covalent protein–protein interactions (Jonas and Kaczmarek, 1996 and Yi et al., 2001). The biophysical characteristics can also be dramatically modified pharmacologically, a fact that serves as the basis for a diverse array of promising therapeutics (Wickenden, 2002).

http://www.sciencedirect.com.ezproxy.hsclib.sunysb.edu/science?_ob=ArticleURL&_udi=B6T36-49PR9Y1-5&_user=334567&_coverDate=11%2F27%2F2003&_fmt=full&_orig=search&_cdi=4938&view=c&_acct=C000017318&_version=1&_urlVersion=0&_userid=334567&md5=2fd40848628da5e0ffe33c8700ec2db1&ref=full
Therefore, the different kinds of K+ channels open in response to different stimuli: a change in the intracellular Ca2+ concentration, the level of certain G-protein subunits in the cell, or the value of the membrane voltage. Underneath this diversity in gating function, K+ channels have diverse structural domains attached in a modular fashion to the conserved pore unit. Ligand-gated K+ channels typically have cytoplasmic or extracellular domains for binding ligands. Voltage-gated K+ channels have integral membrane domains for sensing voltage differences.

an article from nature news

News
Nature Medicine 9, 1099 (2003) doi:10.1038/nm0903-1099

Profile: Kári Stefánsson

Helen Pearson
Reykjavik
Outspoken doctor Kári Stefánsson founded Iceland's deCODE Genetics to use its citizens' genealogy in the hunt for human disease genes. Here he speaks of his passion for medicine and for his homeland—and about courting controversy in both.

In the bowels of the world's most notorious biotech company, a robot, balanced on an earthquake-proof concrete slab, is shuffling the chilled blood of 100,000 Icelanders. It is a stark reminder of the propensity of this desolate island, with its jagged lava fields and steaming fissures, to shudder or explode.

The keeper of the samples is Kári Stefánsson, equally famous for his ability to blow up. One of the most controversial characters in human genetics, the Icelander cofounded deCODE Genetics in 1996 to use the island nation's meticulous genealogy records in his hunt for disease genes. Seven years on, the company has a handful of published results and its stock price is hovering around the bottom of the market.

Stefánsson's background mirrors the history of his volcano-strewn country. He can trace his family back to warrior and poet Egill Skallagrimsson, a first-generation Icelander born in AD 910. Perhaps this heritage imbued Stefánsson with a passion for literature: be it novels, poetry or biography, he always has a book on the go. "You cannot be a good scientist without reading 50−60 novels a year," he says.

Books aside, Stefánsson says his driving force is the desire to help diagnose and cure diseases. After studying medicine in Iceland's capital, Reykjavik, he joined the University of Chicago's neurology and neuropathology faculty, subsequently taking a position at Harvard University in Cambridge, Massachusetts.

It was in a coffee shop in nearby Boston that he and longtime colleague Jeffery Gulcher realized their destiny lay back in Iceland. The pair was already using Icelanders' genealogy to link distantly related people and find the genes they shared for multiple sclerosis risk. If they were to extend their hunt to other common conditions, Stefánsson realized, they needed to live in Iceland. Gulcher was less keen but eventually followed him. "The first thing you tell yourself is that it's an interesting idea but completely impractical," Gulcher now says. "But by and large [Stefánsson] ends up being right."

Stefánsson returned to Iceland to face fierce condemnation of his plans. Much of it centered on the Icelandic parliament's decision to allow deCODE to exclusively build and market a centralized health-care database containing patients' medical records, for which participants are presumed to have given consent. A second criticism has surrounded the commercial use of Icelanders' health information and genetics.

Stefánsson recalls this period as his most difficult. "I felt somewhat persecuted," he says. Outside of the health-care database, he says, blood samples—and hence genetic information—have always been donated with informed consent. In response to the second criticism, he says he could not otherwise have raised the capital, and that Iceland has benefited from the new jobs.

Despite his wounded feelings, Stefánsson shoulders some of the blame for eliciting criticism. One reason is his tendency to lash out or speak provocatively where others would hold their tongue. "He's used his personality to his advantage in creating attention," says friend, collaborator and fellow neurologist Allan Levey of Emory School of Medicine in Atlanta, Georgia. "We know he's earned himself a few enemies."

Some of Stefánsson's fiercest opponents have been those in the Icelandic lobby group Mannvernd. Although the health-care database is still passing security checks and has not yet been built, Mannvernd chairman and psychiatrist Peter Hauksson remains opposed to its construction. He also believes that doctors nowadays are not disclosing their financial interests, such as shares, in deCODE. "They have sold their soul," Hauksson says.

But at least on the surface, many Icelanders seem largely supportive of Stefánsson's plans. Around 100,000 of the nation's 285,000 citizens have given blood and consented to its use in deCODE's genetic studies, and polls show that the majority are in favor of the health-care database. "[Stefánsson] is a very good salesman," concedes Tomas Zoega of National University Hospital in Reykjavik and ex-chair of the Icelandic Medical Association's ethics committee. "He's a colorful personality in Iceland.

"While the controversy rumbles on, Stefánsson and his team have been getting on with some science. In the absence of the notorious health-care database, the company has built up the country's genealogy, other medical records and genetic profiles into three linked databases. When studying a particular disease, collaborating doctors hand their patients' medical information to deCODE, where it is then encrypted and fed into the databases. From this, the researchers can identify those individuals who are even remotely related and ask doctors to approach them for blood samples for genetic analysis. Participants get a deCODE T-shirt.

A personal highlight of his work, Stefánsson says, was the discovery of a gene, dubbed neuregulin-1, implicated in schizophrenia—a condition that afflicts his own brother. "It was a bit of a poetic moment," he says. But though the deCODE team claims to have identified genes linked to conditions from hypertension to aging, the scientific community is yet to be convinced. One key test will be whether the associations between gene and disease hold up in other populations, explains geneticist Pui-Yan Kwok of the University of California, San Francisco. "We're waiting to see whether it actually works," he says.

Stefánsson clearly has no such doubts: he says he hopes to convert the disease genes discoveries into "at least ten" real drugs. By founding deCODE, Stefánsson may have chosen a rocky road on a rocky island in the North Atlantic but, he says, "I wouldn't want to be anywhere else in the world."

Tonight's first speaker: Kári Stefánsson

Dr. Kári Stefánsson, M.D., Dr.Med. from the University of Iceland, is the Chairman, CEO and co-founder of deCODE Genetics and a former professor of neurology, neuropathology and neuroscience at Harvard University (1993-1997). From 1993-1996 he was director of neuropathology at Boston's Beth Israel Hospital. Dr. Kári also held faculty positions at the University of Chicago.

Kári opened the NASDAQ Stock Market on July 20, 2005, after deCode's five year anniversary on the NASDAQ Market.

He was on the Time 100 list in 2007.

(Wikipedia.)

http://stephenslab.uchicago.edu/software.html

3rd speaker
Brendan J. Keating
http://public.nhlbi.nih.gov/GeneticsGenomics/home/care.aspx

What's this?

What's this website about?
http://www.hedweb.com/confile.htm

What's this person doing?
http://david-pearce.com/

CSHL Cliniccal Cardiovascular Genomics meeting

Cannabinoids

Cannabinoids are a group of terpenophenolic compounds present in Cannabis (Cannabis sativa L). The broader definition of cannabinoids refer to a group of substances that are structurally related to tetrahydrocannabinol (THC) or that bind to cannabinoid receptors. The chemical definition encompasses a variety of distinct chemical classes: the classical cannabinoids structurally related to THC, the nonclassical cannabinoids, the aminoalkylindoles, the eicosanoids related to the endocannabinoids, 1,5-diarylpyrazoles, quinolines and arylsulphonamides and additional compounds that do not fall into these standard classes but bind to cannabinoid receptors.[1] The term cannabinoids also refers to a unique group of secondary metabolites found in the cannabis plant, which are responsible for the plant's peculiar pharmacological effects.

Currently, there are three general types of cannabinoids: herbal cannabinoids occur uniquely in the cannabis plant; endogenous cannabinoids are produced in the bodies of humans and other animals; and synthetic cannabinoids are similar compounds produced in a laboratory.


CB1 receptors are found primarily in the brain, specifically in the basal ganglia and in the limbic system, including the hippocampus. They are also found in the cerebellum and in both male and female reproductive systems. CB1 receptors are essentially absent in the medulla oblongata, the part of the brain stem that is responsible for respiratory and cardiovascular functions. Thus, there is not a risk of respiratory or cardiovascular failure as there is with many other drugs. CB1 receptors appear to be responsible for the euphoric and anticonvulsive effects of cannabis.

CB2 receptors are almost exclusively found in the immune system, with the greatest density in the spleen. CB2 receptors appear to be responsible for the anti-inflammatory and possibly other therapeutic effects of cannabis.

THC is the primary psychoactive component of the plant. Medically, it appears to ease moderate pain and to be neuroprotective. THC has approximately equal affinity for the CB1 and CB2 receptors. Its effects are perceived to be more cerebral



Endocannabinoids serve as intercellular 'lipid messengers', signaling molecules that are released from one cell and activate the cannabinoid receptors present on other nearby cells. Although in this intercellular signaling role they are similar to the well-known monoamine neurotransmitters, such as acetylcholine, GABA or dopamine, endocannabinoids differ in numerous ways from them. Neurotransmitters are commonly small, water-soluble molecules that are contained within, and released from, tiny membrane-bound vesicles inside cells. Vesicles are often found in the tips, ‘terminals’, of long cellular branches called axons, and complex morphological and biochemical specializations mark the location from which vesicular release occurs. Endocannabinoids are lipophilic molecules that are not very soluble in water. They are not stored in vesicles, and exist as integral constituents of the membrane bilayers that make up cells. They are believed to be synthesized 'on-demand' rather than made and stored for later use. The mechanisms and enzymes underlying the biosynthesis of endocannabinoids remain elusive and continue to be an area of active research.

Endocannabinoids are described as ‘retrograde’ transmitters because they most commonly travel ‘backwards’ against the usual synaptic transmitter flow. They are in effect released from the postsynaptic cell and act on the presynaptic cell, where the target receptors are densely concentrated on axonal terminals in the zones from which conventional neurotransmitters are released. Activation of cannabinoid receptors temporarily reduces the amount of conventional neurotransmitter released. This endocannabinoid mediated system permits the postsynaptic cell to control its own incoming synaptic traffic. The ultimate effect on the endocannabinoid releasing cell depends on the nature of the conventional transmitter that is being controlled. When the release of the inhibitory transmitter, GABA, is reduced, the net effect is an increase in the excitability of the endocannabinoid-releasing cell. Conversely, when release of the excitatory neurotransmitter, glutamate, is reduced, the net effect is a decrease in the excitability of the endocannabinoid-releasing cell.

Endocannabinoids are hydrophobic molecules. They cannot travel unaided for long distances in the aqueous medium surrounding the cells from which they are released, and therefore act locally on nearby target cells. Hence, although emanating diffusely from their source cells, they have much more restricted spheres of influence than do hormones, which can affect cells throughout the body.

Endocannabinoids constitute a versatile system for affecting neuronal network properties in the nervous system.

The current understanding recognizes the role that endocannabinoids play in almost every major life function in the human body. Cannabinoids act as a bioregulatory mechanism for most life processes, which reveals why medical cannabis has been cited as treatments for many diseases and ailments in anecdotal reports and scientific literature. Some of these ailments include: pain, arthritic conditions, migraine headaches, anxiety, epileptic seizures, insomnia, loss of appetite, GERD (chronic heartburn), nausea, glaucoma, AIDS wasting syndrome, depression, bipolar disorder (particularly depression-manic-normal), multiple sclerosis, menstrual cramps, Parkinson's, trigeminal neuralgia (tic douloureux), high blood pressure, irritable bowel syndrome, and bladder incontinence

Might stimulate some thinking (from the same paper)

Inversion of expression. Specific gene-expression profiles map onto different discharge response types. The most striking example is a near-perfect inversion of the expression profile between cells that discharge initially with a burst onset (b-subtype) and those with a delayed onset (d-subtype)85. So, even cells that are normally classified in the same broad class, such as fast spiking, can have diametrically opposite expression profiles depending on the onset response. This finding also indicates that only a few transcription factors might control the expression of entire sets of ion-channel genes, in which case it is probable that different combinations of transcription factors would give rise to a finite number of distinct electrical classes.

Neuro-circuits & Interneurons

Interestingly, the static (quantal) and dynamic (depression and facilitation) properties of facilitating synapses from single pyramidal neurons onto interneurons vary across layers126, which might cause targeted inhibitory cells in deep cortical layers to discharge before those in supragranular layers. Such layer-specific differences in the recruitment of interneurons could influence the direction of information flow in the cortical column.


Remarkably, all the synapses formed by one interneuron onto multiple pyramidal neurons show identical synaptic dynamics15. All the synapses from an interneuron onto all targets of the same type (pyramidal neurons in this case) seem to have identical release probabilities and time constants for recovery from synaptic depression and facilitation. This homogeneity principle contrasts sharply with the heterogeneity of glutamatergic synapses formed by a pyramidal neuron onto other pyramidal neurons and also has implications for the forms of learning that might shape these synapses. The absolute strength of these synaptic connections is heterogeneous (probably due to different numbers of synapses and/or postsynaptic receptors), indicating that they could be modified by the relative timing of activity in only one pair of neurons (presynaptic and postsynaptic), but their dynamics are homogeneous, suggesting that these parameters must be modified by the activity patterns of the entire population of postsynaptic pyramidal neurons relative to the single presynaptic interneuron.

Why balance Yang with Yin? Why does excitation need to be balanced with inhibition and why do transient moments of imbalance occur? This is a vast area, which will not be dealt with in this review, except to speculate on two potential reasons. At the level of individual neurons, matching inhibition as a function of stimulus intensity could allow information to be processed and encoded at a higher or lower temporal resolution, depending on the baseline firing rates. This can be achieved by changing the membrane time constant, which changes the time window for temporal integration161 (see also Ref. 162) and by changing the temporal precision of spike generation by adding high-frequency membrane 'noise'163, 164, 165. At this level, balance might be required to normalize the baseline for synaptic integration as a function of activity (to normalize the mutual information between the input channels) and spiking might reflect moments of imbalance (high mutual information between the input channels). At the microcircuit level, a sliding scale between integration and coincidence detection as a function of activity in each neuron could be important to control which neurons synchronize at which frequencies162. Balance might be required to keep all neurons independent (to normalize mutual information across neurons) and oscillations might reflect orchestrated momentary imbalances of groups of neurons (high mutual information between neurons). Needless to say, considerable work is required to test and turn theory into fact.

http://www.nature.com.ezproxy.hsclib.sunysb.edu/nrn/journal/v5/n10/full/nrn1519.html

Kv3 channels

Analysis of the Kv3 subfamily of K+ channel subunits has lead to the discovery of a new class of neuronal voltage-gated K+ channels characterized by positively shifted voltage dependencies and very fast deactivation rates. These properties are adaptations that allow these channels to produce currents that can specifically enable fast repolarization of action potentials without compromising spike initiation or height. The short spike duration and the rapid deactivation of the Kv3 currents after spike repolarization maximize the quick recovery of resting conditions after an action potential.

The ability to fire action potentials at high frequencies (often up to 1 kHz) or to follow high-frequency inputs are important physiological functions of numerous cells throughout the mammalian CNS. Neuronal populations within such disparate regions as the hippocampus, basal ganglia, neocortex, reticular thalamus, medial vestibular nucleus and auditory nuclei are capable of responding to afferent input with action potentials of brief duration and of firing repetitively at high frequencies.

Although numerous channel types have been implicated in conferring such properties, the voltage-gated K+ channels of the Kv3 subfamily have now been identified as major determinants of the FS phenotype.

Molecular characteristics of Kv3 subfamily members
Both rodents and humans possess four Kv3 genes: Kv3.1, Kv3.2, Kv3.3 and Kv3.4 ([6 and 7]). All four Kv3 genes generate multiple protein isoforms by alternative splicing, which produces versions with different intracellular C-terminal sequences. There are now 13 different Kv3 proteins known in mammals (Kv3.1a–Kv3.1b, Kv3.2a–Kv3.2d, Kv3.3a–Kv3.3d and Kv3.4a–Kv3.4c), yet the currents expressed in heterologous expression systems by the spliced isoforms of each Kv3 gene are virtually indistinguishable. Recent studies suggest that the alternatively spliced C termini confer isoform-specific regulation by second messenger signaling systems and targeting to distinct neuronal compartments.

Nature of Random, Random of nature

What's nature? The property innate in it, is something called radom.

Through infinite length of time, it created the ball like thing, stars. It create a network of these stars. And all the things it made up make up itself, nature. All this things take place like a miracle, but you can still imagine them happen.

But there's something hard to believe, is these random nature can create living things. It has no powful tools, except for using two things: randomness, and time. And today, when we, human beings, the highest achievement of nature, with our somewhat intelligant brains, coming to try to unravel the mechanisms produced by random, still totally get lost. Assembly of random happenings, v.s. those intelligent scientific brains, the biggest joke hah? But still, the latter still striving hard.

Nature depends on time to evolve. But we need to compete against time.

Meeting Buzsaki

So, how does everything goes with Gyorgy Buzsaki?

Well, mixed feelings.

This is one of the greatest scientific talk that I have ever experienced. The whole conference room is silence, entire silence. The talk is interesting, not because of showing some fantastic, funny while amazing things, but by the way it's clearly shown. The speaker just draw you into the deep world of his logical world. You can't deny anything because everything is so convincing, and because you just want to get into the new insight he opened to you try to imagine further. I'm just wondering what he can do in his next talk if he want to use different slides, because this series of slide is almost perfect. Perfectly clear and reasonable.

You might imagine the question section to be even hotter than last time's. Actually no. Paul Adams is also there, but he didn't question anything. Unusualy, this time's faculty tend to be silent, while students asked some questions. You know in normal times, the faculties came up with a lot of brilliant questions. But this time, few of them asked, and actually the ones even I can come up with an answer.

David say he seems not welcoming questions. Well, think over, that's seems true. Most of the seminar speakers I see, will specifically encourage people to ask and don't forget to give feedback to the questions they asked. He didn't do that specially. I'm sure he likes people asking questions. But the lecture he made, seems to give no chance for people to ask questions. I still remember when we went to talk with him about some questions after the talk, he would tell us that there's foible where where on his own.

Actually I didn't ask any questions except for asking if there are any way to get an discount on his book. :) Everyone got into laugh including him. But he did answer my questions seriously. Normally I asked a lot of questions during the one and a half hour discussion, but you know what, this time is mainly he asking us questions! He asked us why choose neuroscience rather any other scientific subjects, why neuroscience rather that other biology branches; what's the difference between people in biotechnology and us. He said I'm sure none of you come to this field aim to publish a paper. Silence this time, because everyone can't imagine how you can get graduated without publishments, how we can get our future jobs without a paper. Then he said do any one of you worry about what kind of position you'll get later, we don't care, right? Silence again, of course you don't need to worry about anything like this. But you are true, when we just choose this field, we don't think about papers, tenures -- only science.

All the questions he asked I never gave out the one he wanted. He gave an example that imagine there's 10billion treasure underneath somewhere of SB campus, what's the quickest way to get it. I said buy the whole campus then you can do whatever you want. Of course I mistook him, he is looking for a scientific answer. The securest way is to dig the campus square by square, but the quickest way is just random search. All through the whole lunch time is like this, he raised many questions, we are like headless flies, try to think and answer as much as possible but never got his clue.

The heart v.s. the brain

One question I want to ask Gyorgy Buzsaki:
When we look at the wiring mechanism of neurons, is the heart comparable to the brain in some way?

Both in the heart and the brain, there're electrical events going on, there's resting potential and depolarization, there're similarly functioning ion channels. Oh, gap junctions, that's special in the heart. One difference is that, the target of the electrical signals are actually contented inside the heart, i.e., the whole events from stimulation to response, happens in the same organ. In contrast, it's more likely that the brain gives signal, and the outside receptors response.

But when we consider the electrical wiring of the brain, is it possible to use the heart as a simple model? You know, many ion channelists favor to choose the heart as the target organ for research, which contain simliarly functioning ion channels, but much easier for them to unravel the mechanisms underlying.

Or, given the gap junction thing, no way?

Well, I need to think more before I went up to ask him.

Janelia Farm

When I just came to CSHL, there're already several established scientists ruthlessly leaving the beautiful place. Where did they go? Janelia Farm. The post docs are so surprised when finding that I've no idea about this Holy Land of biomedical research. They can't help to describe how cool it is, which to me, was like a legend.

No wonder the most alluring thing is the full support from HHMI. You keep a small lab, you just need to stay in the lab, focusing on your experiments and search peer papers. No need to worry about funding, no need to worry about how can I my postdoc. You just need to do your science, pure science, completely science! And the transparent structure of the building construction, you see it? I love that! I have stayed in the department office for 1 year, you know how it's like, no windows, the only outlet is the door, small room, all walls surrouding, no difference in day and night, dead silence if you close the door and shut down all the machines, you know what's that like? A jail.

Oh, there's a 5 minutes documentation available.

http://www.hhmi.org/janelia/discover_janelia/index2.html

Description about Jenelia Farm:

We aim to identify important biomedical problems for which future progress requires technological innovation and then we foster the establishment of integrated teams of biologists and tool builders who seek to break through existing barriers.

two synergistic areas that are particularly well matched to the Janelia Farm environment:
1. The identification of general principles that govern how information is processed by neuronal circuits
2. The development of imaging technologies and computational methods for image analysis

Graduate Program:

The Howard Hughes Medical Institute offers two programs leading to the Ph.D. based at its Janelia Farm Research Campus, in partnership with the universities of Cambridge and Chicago. These accelerated programs are designed for a small number of well-prepared, highly committed, and gifted students. We offer opportunities for interdisciplinary research in an intense environment.

People from CSHL:

Karel Svoboda

The marks building becomes so empty as he left. The good thing is the whole floor become Tony and Zach's. When I rotate there, I was always the only one on the first floor which originally was his lab. Heard of a lot about him, he is a very positive, active, and creative scientist, in everyone's eys.

Dmitri B. Chklovskii

I remember he do computational neuroscience.

Alla Y. Karpova

It surprised me so much when finding that she came here. Actually in marks building, her bench is just opposite to my bench. Funny enough that I met her first when I was doing experiments in one midnight, she gave me a shock when she appeared and went by quickly. Later I joined the farewell party for her.

Gyorgy Buzsaki

http://osiris.rutgers.edu/BuzsakiHP/Personnel/Members/Buzsaki/buzsaki.html He must be a very interesting guy, I'm looking forward to meeting him tomorrow!

The way to understand learning and memory

In György Buzsáki's 2003 nature paper, he proposed that except for spatial prediction, firing pattern of peer cells will add to the accuracy of the prediction. They found that neurons get sychronized with their peer cells, forming cell assemblies. Basically these cells have a preference to while cell assemblies to join in, in turn you can actually catergorize the other cells into two: positive-weighted cells (cells that this neuron will always join to fire), negative-weighted cells(cells that this neuron don't join to fire).

The paper also related EEG to the predicability. I don't know how the EEG will contribute to the firing possibility or how much the firing of this neuron will change EEG. But it seems that cells only fire at the trough of theta wave. So to my knowledge, theta wave is the collective activity of the brain neurons or hippocampal neurons, which to some degree, can be understood as the background neural activity. Does the firing lying at the trough phase of the theta wave means distinct from background activity, which in turn can be dictinct and received by the subordinate neurons?

Also after reading and discussing this paper, I can draw a propose about how to understand learning and memory. Well, this in the end actually turned out to be the general thing everyone is trying to look up, but still, I'd like to describe it here:
Neurons tend to fire in assembly, or the firing of neurons tend to be sychronized or dissynchronized by peer cells. When several different sensory inputs get into the brain, different sets of neurons get fired. At first there's nothing specific happening between this set of neuron and that set. But when this simultaneous happening came up again, neurons from different sets are kind of getting related, or associated. With repeats, the synchronization set up. That's associative learning. If we try to understand in the molecular or cellular level, that is, the synchronization made the originally weak synapses or silent synapses got strengthened (namely LTP?), which vice versa, the dissynchronization make the originally strong synapses weaker (namely LTD?).

The good thing we assume here is that neurons tend to set up synapses with any other neurons. They lie there, do something, or not doing anything. Frequent electrical signals make them strengthened. When no electric signals coming to knock at their door, they just lie there silently. They are ready to go whenever the electrical order come.

But what about a different propose here? Synapses assemble in response to the need and disassemble when no longer needed. So how can synapses between different sets of neurons set up? Think about this, the electric signal go through the whole neuronal processes, which elicit chemical/physical changes in the processes. Once both the two neighboring neurons undergo the chemical/physical changes, synapse tend to form between them.

Well, I can go on and go on, become a total theorist. I need to go to read more literatures to support or dissupport my idea. (Only if someone can favor me more time.)

The 20ms in Neuroscience?

Last week in Steve's seminar, when proposing the non-Hebbian ISDP, he said 20ms seems to be special for the paring of Perforant pathway and Schaffer Colateral Pathway. It's kind of strange that 20ms to be a special number, as I can tell from his own feeling.

Today, I'm reading this 2003 nature paper about hippocampal cell assembly. They propose 25ms to be the best timescale for sychrony. And they think this to be physiologically significant in 3 ways: 1. it matches the membrane time constant for pyramidal neurons in hippocampus. 2. it matches the period of the hippocampal gamma oscillation. 3. it matches the effective window for synaptic plasticity.

So, 20-25 ms, the lucky number in neuroscience?

Oscillators--- Neuronal Oscillation

Harmonic oscillator 简谐子！简谐运动！
F=-kx

Relaxation oscillator

A relaxation oscillator is an oscillator in which a capacitor is charged gradually and then discharged rapidly. The capacitor is charged through the resistor, causing the voltage across the capacitor to approach the charging voltage on an exponential curve. In parallel with the capacitor is the threshold device. Such devices don't conduct at all until the voltage across them reaches some threshold (trigger) voltage. They then conduct heavily, quickly discharging the capacitor. When the voltage across the capacitor drops to some lower threshold voltage, the device stops conducting and the capacitor can begin charging again, repeating the cycle. If the threshold element is a neon lamp, the circuit also provides a flash of light with each discharge of the capacitor.

Steven A. Siegelbaum, HHMI investigator

Introduction in HHMI

Dr. Siegelbaum is also Professor of Pharmacology in the Center for Neurobiology and Behavior at Columbia University College of Physicians and Surgeons. He received his A.B. degree in biochemical sciences from Harvard College and his Ph.D. degree from Yale University, where he studied the role of calcium in cardiac electrical activity. He then did postdoctoral research with David Colquhoun at University College London and with Philippe Ascher at the Ecole Normale Supérieure in Paris, studying the nicotinic acetylcholine receptor ion channel. Dr. Siegelbaum has received the Herbert J. Kayden Award in biomedical science from the New York Academy of Sciences.

Last Thursday our program invited Dr. Siegelbaum over to give a talk. Before hand we read the pre-print handed out by him, it's the 2004 paper in cell. Actually this paper elicited strong discussion in our class, although a little bit negative to it. It's normal because it's really hard when things suddenly comes to behavior level, every result significant or unsignificant, hard to believe, expecially for molecularists. It's hard to deny that this paper can go to Cell probably partly because of the biggest people in neuroscience, Kandel. He is a extreme character in scientific world, as David said.

When I first saw Steve on the seminar, he is like the picture above, looks very nice as David told us. Actually I heard his voice when he came to David's office and I was just in the lab doing experiments. I like the way he walked slowly in the front while speaking, with the look of thinking deeply. That's the scientist, I said to myself. He started from the background of cerebellum, then come the HCN channels and the figures from the paper, not staying there too long, he went to his non-Hebbian thoery---Input Timing-Dependent Plasticity(ITDP). The question section was really hot. Paul Adams was also there. Of course he will definitely be the core listener. One of his questions is actully mine. :( :( Like always, later on I felt very regretted for being not confident enough to ask questions.

Later we eat lunch together. That's a very nice time. I planned to wrote something about him. But coming up so late, I could simply wrote a diary, forgetting all those words I kept in mind to say.

Old knowledge, but worth to save

碱裂解法抽提质粒原理

http://bbs.bioon.com/dispbbs.asp?BoardID=95&ID=85175&replyID=&skin=1

碱裂解法从大肠杆菌制备质粒，是从事分子生物学研究的实验室每天都要用的常规技术。为了方便理解，这里罗列一下碱法质粒抽提用到三种溶液： 溶液I，50 mM葡萄糖 /25 mM Tris-Cl / 10 mM EDTA，pH 8.0； 溶液II，0.2 N NaOH / 1% SDS； 溶液III，3M 醋酸钾 / 2 M 醋酸。

让我们先来看看溶液I的作用。任何生物化学反应，首先要控制好溶液的pH，因此用适当浓度的和适当pH值的Tris-Cl溶液，是再自然不过的了。那么50mM葡萄糖是干什么的呢？说起来不可思议，★加了葡萄糖后最大的好处只是悬浮后的大肠杆菌不会快速沉积到管子的底部。因此，如果溶液I中缺了葡萄糖其实对质粒的抽提本身而言，几乎没有任何影响。所以说溶液I中葡萄糖是可缺的。 那么EDTA呢？大家知道EDTA是Ca2+和Mg2+等二价金属离子的螯合剂，配在分子生物学试剂中的主要作用是：★抑制DNase的活性，和抑制微生物生长。在溶液I中加入高达 10 mM 的EDTA，无非就是要把大肠杆菌细胞中的所有二价金属离子都螯合掉。如果不加EDTA，其实也没什么大不了的，只要不磨洋工，只要是在不太长的时间里完成质粒抽提，就不用怕DNA会迅速被降解，因为最终溶解质粒的TE缓冲液中有EDTA。如果哪天你手上正好缺了溶液I，可不可以抽提质粒呢？实话告诉你，只要用等体积的水，或LB培养基来悬浮菌体就可以了。有一点不能忘的是，菌体一定要悬浮均匀，不能有结块。

轮到溶液II了。这是用新鲜的0.4 N的NaOH和2％的SDS等体积混合后使用的。要新从浓NaOH稀释制备0.4N的NaOH，无非是为了保证NaOH没有吸收空气中的CO2而减弱了碱性。很多人不知道其实破细胞的主要是碱，而不是SDS，所以才叫碱法抽提。★事实上NaOH是最佳的溶解细胞的试剂，不管是大肠杆菌还是哺乳动物细胞，碰到了碱都会几乎在瞬间就溶解，这是由于细胞膜发生了从bilayer（双层膜）结构向micelle（微囊）结构的相变化所导致。用了不新鲜的0.4 N NaOH，即便是有SDS也无法有效溶解大肠杆菌（不妨可以自己试一下），自然就难高效率抽提得到质粒。如果只用SDS当然也能抽提得到少量质粒，因为SDS也是碱性的，只是弱了点而已。很多人对NaOH的作用误以为是为了让基因组DNA变性，以便沉淀，这是由于没有正确理解一些书上的有关DNA变性复性的描述所导致。有人不禁要问，既然是NaOH溶解的细胞，那为什么要加SDS呢？那是为下一步操作做的铺垫。这一步要记住两点：第一，时间不能过长，千万不要这时候去接电话，因为在这样的碱性条件下基因组DN□断会慢慢断裂；第二，必须温柔混合（象对待女孩子一样），不然基因组DNA也会断裂。基因组DNA的断裂会带来麻烦，后面我再详细说明。

每个人都知道，溶液III加入后就会有大量的沉淀，但大部分人却不明白这沉淀的本质。最容易产生的误解是，当SDS碰到酸性后发生的沉淀。如果你这样怀疑，往1%的SDS溶液中加如2M的醋酸溶液看看就知道不是这么回事了。大量沉淀的出现，显然与SDS的加入有关系。如果在溶液II中不加SDS会怎样呢，也会有少量的沉淀，但量上要少得多，显然是盐析和酸变性沉淀出来的蛋白质。既然SDS不是遇酸发生的沉淀，那会不会是遇盐发生的沉淀呢？在1％的SDS溶液中慢慢加入5 N的NaCl，你会发现SDS在高盐浓度下是会产生沉淀的。因此高浓度的盐导致了SDS的沉淀。 但如果你加入的不是NaCl而是KCl，你会发现沉淀的量要多的多。这其实是十二烷基硫酸钠（sodium dodecyl sulfate）遇到钾离子后变成了十二烷基硫酸钾（potassium dodecylsulfate,PDS），而PDS是水不溶的，因此发生了沉淀。如此看来，★溶液III加入后的沉淀实际上是钾离子置换了SDS中的纳离子形成了不溶性的PDS，而高浓度的盐，使得沉淀更完全。大家知道SDS专门喜欢和蛋白质结合，平均两个氨基酸上结合一个SDS分子，钾钠离子置换所产生的大量沉淀自然就将绝大部分蛋白质沉淀了，让人高兴的是大肠杆菌的基因组DNA也一起被共沉淀了。这个过程不难想象，因为基因组DNA太长了，长长的DNA自然容易被PDS给共沉淀了，尽管SDS并不与DNA分子结合。 那么2M的醋酸又是为什么而加的呢？★是为了中和NaOH，因为长时间的碱性条件会打断DNA，所以要中和之。基因组DNA一旦发生断裂，只要是50－100 kb大小的片断，就没有办法再被PDS共沉淀了。所以碱处理的时间要短，而且不得激烈振荡，不然最后得到的质粒上总会有大量的基因组DNA混入，琼脂糖电泳可以观察到一条浓浓的总DNA条带。 很多人误认为是溶液III加入后基因组DNA无法快速复性就被沉淀了，这是天大的误会，因为变性的也好复性的也好，DNA分子在中性溶液中都是溶解的。NaOH本来是为了溶解细胞而用的，DNA分子的变性其实是个副产物，与它是不是沉淀下来其实没有关系。溶III加入并混合均匀后在冰上放置，目的是为了PDS沉淀更充分一点。不要以为PDS沉淀的形成就能将所有的蛋白质沉淀了，★其实还有很多蛋白质不能被沉淀，因此要用酚/氯仿/异戊醇进行抽提，然后进行酒精沉淀才能得到质量稳定的质粒DNA，不然时间一长就会因为混入的DNase而发生降解。

这里用25/24/1的酚/氯仿/异戊醇是有很多道理的，这里做个全面的介绍。 ★酚（Phenol）对蛋白质的变性作用远大于氯仿，按道理应该用酚来最大程度将蛋白质抽提掉，但是水饱和酚的比重略比水重，碰到高浓度的盐溶液（比如4M的异硫氰酸胍），离心后酚相会跑到上层，不利于含质粒的水相的回收；★但加入氯仿后可以增加比重，使得酚/氯仿始终在下层，方便水相的回收；★还有一点，酚与水有很大的互溶性，如果单独用酚抽提后会有大量的酚溶解到水相中，而酚会抑制很多酶反应（比如限制性酶切反应），应此如果单独用酚抽提后一定要用氯仿抽提一次将水相中的酚去除，而用酚/氯仿的混合液进行抽提，跑到水相中的酚则少得多，★微量的酚在乙醇沉淀时就会被除干净而不必担心酶切等反应不能正常进行。★至于异戊醇的添加，其作用主要是为了让离心后上下层的界面更加清晰，也方便了水相的回收。 ★回收后的水相含有足够多的盐，因此只要加入2倍体积的乙醇，在室温放置几分钟后离心就可以将质粒DNA沉淀出来。这时候如果放到-20℃，时间一长反而会导致大量盐的沉淀，这点不同于普通的DNA酒精沉淀回收，所以不要过分小心了。高浓度的盐会水合大量的水分子，因DNA分子之间就容易形成氢键而发生沉淀。如果沉淀，就用70％的乙醇多洗几次，每次在室温放置一个小时以上，并用tip将沉淀打碎，就能得到好的样品。得到的质粒样品一般用含RNase（50ug/ml）的TE缓冲液进行溶解，不然大量未降解的RNA会干扰电泳结果的。琼脂糖电泳进行鉴定质粒DNA时，多数情况下你能看到三条带，但千万不要认为你看到的是超螺旋、线性和开环这三条带。碱法抽提得到质粒样品中不含线性DNA，不信的话你用EcoRI来线性化质粒后再进行琼脂糖电泳，就会看到线性质粒DNA的位置与这三条带的位置不一样。其实这三条带以电泳速度的快慢而排序，分别是超螺旋、开环和复制中间体（即没有复制完全的两个质粒连在了一起）。如果你不小心在溶液II加入后过度振荡，会有第四条带，这条带泳动得较慢，远离这三条带，是20-100kb的大肠杆菌基因组DNA的片断。

In-vivo Electroporation

Through out my third rotation in CSHL, I made several constructs first, then tried in-vivo electroporation and finally successed. I did molecular myself, but this complicate experiment is done after watching the old technician doing this several times. He showed me this website, which I just came up and reminded me of writing something.
http://www.m.chiba-u.ac.jp/class/dev/in_vivo_electroporation.html

Weirdly, he can't get the GFP expressed in the brain using this methods. We just thought there're some problems: 1. CMV promoter is not good for electroporation. 2. Our techniques are not mature. He tried twice as many rats as me (I don't have time that much because of having classes and TA in stony brook). But you know what, at the last week of my rotation, the last mother rat give births, I did found that I got expression on the day before I leave!

Finally there is no regret in that rotation. I wrote a protocol and emailed the results altogether to the professor. I know he like me. Actually he feels very sorry of not being able to take me.

How to avoid Satellite colonies

Although I think satellite colonies don't bother. But I do found some knowledge useful.

Quoted from bioforum:

Ampicillin plates (100ug/mL) often show satellite colonies after incubating for more than 16 hours (my plates show satellites after 20 hours incubation). So it is better to incubate for a shorter period.

Use methicillin/ampicillin. I think it is available from Sigma. Bacteria that can grow in ampicillin can grow in met/amp, but you don't get satellite colonies.

We had the same problem and switched to carbenicillin, while using the same vecotr, and no more satellites but colony growth.

You could also try using carbenicillin instead of ampicillin. Carbenicillin is more stable than ampicillin and so would be effective for a longer period of time. It is, however, very much more expensive.

HCN channels: Hyperpolarization-activated cation currents

Hyperpolarization-activated cation currents, termed If, Ih, or Iq, were initially discovered in heart and nerve cells over 20 years ago. These currents contribute to a wide range of physiological functions, including cardiac and neuronal pacemaker activity, the setting of resting potentials, input conductance and length constants, and dendritic integration. The hyperpolarization-activated, cation nonselective (HCN) gene family encodes the channels that underlie Ih.

Unlike most voltage-gated channels, Ih channels are activated by hyperpolarizing voltage steps to potentials negative to −60 mV, near the resting potentials of most cells. This property earned them the designation of If for “funny” or IQ for “queer”

Ih is perhaps most widely known for its proposed role in the generation of spontaneous pacemaker activity, both in the heart and central nervous system. As a result, Ih is often referred to as the pacemaker current. However, it is clear that the spontaneous firing of certain cells, such as Purkinje neurons in the cerebellum and respiratory neurons in the brainstem , do not require the participation of Ih to generate automaticity. Even in the heart, the importance of Ih as the prime generator of depolarizing pacemaker current has been questioned.

Noma & Irisawa first reported the existence in sino-atrial node (SAN) tissue of a slow, time-dependent inward current that was activated by membrane hyperpolarization, later termed Ih. DiFrancesco and colleagues first provided a detailed characterization of this current, which they named If. Shortly thereafter, a similar hyperpolarization-activated cation current was identified in rod photoreceptors and hippocampal pyramidal neurons, which was termed IQ.

Ih is a mixed cation current that typically activates with hyperpolarizing steps to potentials negative to −50 to −60 mV. The kinetics of activation during a hyperpolarization, and deactivation following repolarization, are complex. Activation is usually preceded by a significant delay, resulting in a marked sigmoidal time course of onset for Ih. Following this delay, channel opening can be empirically described by either a single or double exponential function, depending on the cell type. These exponential kinetics also vary widely among different cells. In heart and thalamic relay neurons, the time course of activation is quite slow, requiring several seconds to reach a steady state. In hippocampal CA1 neurons by contrast, the kinetics of activation are quite rapid, with time constants of activation on the order of 30 to 60 ms.

The Ih channel has unusual ion selectivity in that it conducts both Na+ and K+ ions but excludes Li+. Divalent cations neither permeate nor block the channel. The ratio of the K+ to Na+ permeability of the channel, PK:PNa, ranges from 3:1 to 5:1, yielding values for the reversal potential of −25 to −40 mV. As a result, activation of the channel at typical resting potentials results in a net inward current carried largely by Na+, which will depolarize the membrane toward threshold for firing an action potential. One other unusual feature of the channel is that its conductance is highly sensitive to external K+ levels. Reduction of K+ below normal extracellular levels can result in a dramatic decrease in current magnitude. Ih channels also have a very small single channel conductance. Even in elevated external K+, channel conductance is only 1 pS. The sensitivity of Ih to external K+ might provide an important means for regulating Ih function; for example, external K+ elevation during seizure activity or cardiac ischemia might enhance the magnitude of Ih and thus alter excitability.

The characterization of Ih has often been hampered by its relatively small magnitude, combined with the presence of overlapping ionic currents that activate over a similar range of potentials. These currents include inward rectifier K+ currents, persistent voltage-gated Na+ currents (INaP), hyperpolarization-activated Cl currents, and transient A-type K+ currents. Ih has often been distinguished from these other currents by its sensitivity to relatively low concentrations of external Cs+, which produce substantial (>50%) blockade at a concentration of 1–2 mM, and by its insensitivity to 1–2 mM external Ba2+, a potent blocker of inward rectifier K+ channels. However, Cs+ has the drawback of blocking certain K+ channels at this concentration. A number of organic compounds have been described that block Ih fairly specifically. These include ZD-7288, UL-FS49 (zatebradine), and S-16257 (ivabradine), although zatebradine also blocks K+ channels, and ZD-7288 and DK-AH 269 (which is structurally similar to zatebradine) alter synaptic transmission, independently of blocking Ih.


One of the most interesting and important characteristics of Ih is its regulation by cyclic nucleotides. Neurotransmitters that elevate cAMP levels greatly facilitate the activation of Ih by shifting its voltage dependence of gating to more positive potentials, typically by 10 mV or more. As a result, during a hyperpolarizing step to a given voltage, Ih activates more completely and more rapidly. Conversely, neurotransmitters that downregulate cAMP depress the activation of Ih, shifting its activation curve to more negative potentials. DiFrancesco & Tortora made the surprising discovery that the regulation of Ih by cAMP does not require protein phosphorylation. Rather, because cAMP enhanced channel opening in cell-free membrane patches in the absence of MgATP, DiFrancesco & Tortora concluded that Ih gating is directly regulated by cAMP binding.

The regulation of Ih through cyclic nucleotides contributes to the speeding of the heart rate in response to β-adrenergic agonists (which raise levels of cAMP) and to the slowing of the heart rate in response to muscarinic acetylcholine receptor agonists (which reduce levels of cAMP). In the brain, a number of transmitters have been shown to regulate Ih in different neurons through either enhancing or diminishing cAMP levels. Ih can also be regulated in both brain and heart by nitric oxide, which stimulates soluble guanylate cyclase and elevates cGMP levels. Subsequent studies have suggested that the activity of Ih may also be regulated by protein phosphorylation and dephosphorylation. A number of protein kinases (PKs) have been implicated, including PKA, PKC and tyrosine kinases. However, direct evidence for Ih channel phosphorylation is so far lacking.

PHYSIOLOGICAL ROLE OF Ih: GENERAL PRINCIPLES
At least four physiological roles have been ascribed to Ih: (a) control of pacemaker activity (in both heart and brain), (b) control and limitation of resting potential, (c) control of membrane resistance and dendritic integration, and (d) regulation of synaptic transmission.

In a cell at rest, tonic activation of Ih helps set the level of resting potential at a somewhat depolarized level. In addition, activation of Ih also contributes to the resting membrane conductance. Thus the presence of Ih tends to decrease a cell's input resistance, membrane time constant, and length constant.

MOLECULAR BASIS FOR Ih IN BRAIN

All four HCN isoforms are expressed in the mammalian brain. Of these four, HCN3 shows the weakest expression. HCN2 shows a broad pattern of strong mRNA expression and is present in most brain regions. HCN1 is more selectively expressed. For example, it is prominent in layer 5 pyramidal neurons of the neocortex, but not in other cortical layers. This mRNA expression pattern is consistent with the high density of Ih current recorded in the layer 5 neurons.

HCN expression patterns have been particularly well characterized in the hippocampus. There, HCN1 is found in CA1 and CA3 pyramidal neurons, with somewhat higher levels of expression in CA1 neurons compared with that in CA3 neurons. HCN2, in contrast, is expressed at somewhat higher levels in CA3 than in CA1 pyramidal neurons. HCN1 and HCN2 are also expressed in scattered neurons in the stratum oriens and stratum lucidum regions of the hippocampus. Such cells most likely represent inhibitory interneurons, which have been shown to contain Ih. Expression of HCN1 in the dentate gyrus is quite low, especially in the mouse brain. HCN4 is only weakly expressed in hippocampus and neocortex.

HCN1 is also strongly expressed in the inhibitory basket cells and Purkinje neurons of the cerebellum. In the thalamus, high levels of expression of HCN2 and HCN4 are found in the excitatory thalamocortical relay neurons, whereas only HCN2 is found in the inhibitory thalamic reticular neurons.

MOLECULAR BASIS FOR Ih IN HEART

HCN1, HCN2, and HCN4 are expressed in heart. Their relative mRNA abundance varies with cardiac region, species, age, and, perhaps, disease state. Although there is good correlation between absolute level of HCN message and magnitude of measured Ih among cardiac regions and species, it has proven difficult to explain functional heterogeneity in the heart with specific isoform expression patterns. In this regard, heart differs from brain in that cardiac Ih exhibits a much wider range of regional voltage dependence.

The SAN, the normal pacemaking region of the heart, exhibits both the largest and most positively activating pacemaker current and expresses the highest message level. HCN4 is the most predominant isoform, accounting for >80% of the total HCN mRNA. Significant levels of HCN1 are present in rabbit SAN (20% of total HCN mRNA) but only very low levels are detected in mouse. In dog SAN, low levels of HCN1 (7%) and HCN2 (8%) mRNA make up the remainder of HCN transcripts.

HCN4 is also the predominant HCN transcript in cardiac Purkinje fibers, specialized conducting tissue that can exhibit subsidiary pacemaker activity. Canine Purkinje fibers, which exhibit significant automaticity and display a large Ih, show the highest level of HCN mRNA expression in the heart outside of the SAN (35% of SAN). HCN4 accounts for 90% of the transcripts, with the remainder contributed by HCN2. In contrast, rabbit Purkinje fibers, which tend not to be automatic and exhibit little Ih, show minimal levels of HCN message (4% of SAN). The HCN mRNA that is present represents roughly equivalent levels of HCN1 and HCN4, with a minor contribution of HCN2 (10%).

In the rabbit ventricle only HCN2 is detected; however, overall levels are extremely low and no measurable Ih is found. Canine and rat ventricle exhibit greater Ih and higher HCN levels than rabbit. HCN2 is by far the predominant ventricular isoform, especially in adult animals, with the balance being HCN4.

The distinct biophysical properties of the HCN isoforms expressed in different regions of the heart cannot account for the marked differences in the time-dependence and voltage-dependence of activation of native Ih in these same regions. Thus, although Ih in the SAN activates more rapidly than Ih in other cardiac regions, the predominant isoform expressed in SAN, HCN4, is the most slowly activating isoform in heterologous expression systems. Moreover, whereas the V½ values of the four HCN isoforms vary by no more than 20 mV, the V½ values of native Ih currents may vary by as much as 80 mV between SAN and ventricle. During development, the voltage dependence of Ih activation in ventricular muscle shifts by up to 40 mV toward more negative potentials, so that in adults the threshold for Ih activation is negative to the resting potential [although there is some debate as to whether the developmental change is a shift in voltage dependence or a reduction in current magnitude ]. These results suggest that factors within a cardiac cell can influence the voltage dependence of an individual HCN isoform. In fact, when HCN2 is overexpressed in neonatal and adult rat ventricular myocytes, the voltage dependence is more positive in the neonatal cells, and this difference is independent of basal cAMP levels.


Although the biophysical properties of heterologously expressed HCN isoforms cannot fully account for the observed variation in native Ih, there is some correlation between which isoforms are expressed in a specific region and the voltage dependence of the native pacemaker current; regions with the most negative activation (e.g., ventricle) tend to express HCN2 predominantly, whereas regions with more positive voltage ranges of activation express HCN4. In addition, whereas HCN2 is the dominant ventricular isoform throughout development, the relative expression ratio of HCN2:HCN4 increases from 5:1 in the neonatal rat ventricle to 13:1 in the adult rat ventricle at the same time that the voltage dependence of the native Ih is becoming more negative . The large phenotypic variation in Ih throughout the heart may reflect the differential modulation of HCN subunits by factors such as phosphorylation or auxiliary subunits, which may be turned on in distinct regions or at different developmental stages by the action of hormones, transmitters, or growth factors. Thus the rationale for the regional patterns of HCN isoform expression might be a differential susceptibility of these isoforms to such modulatory changes. In this respect, it is interesting that in a β2-adrenergic overexpressing mouse heart, ventricular HCN4 message is upregulated with no change in HCN2.

http://arjournals.annualreviews.org/doi/full/10.1146/annurev.physiol.65.092101.142734?cookieSet=1

Hippocampus

Paul Adams

I met him in class. He is a real scientist. You have to like him, he was so cute when discribing science to you; You have to admire him, becase you can feel that he is the embodiment of science; You will be moved by the way he dipicts science, you have to be attracted; You feel like you are so far away from him, because all his thoughts are so brilliant that normal people can never easily understand; You want to fall into laugh when he seems to care nothing except for science, the way he wear, the way he drink or move, but you won't because you respect him from the bottom of your heart; He will use any thing surround as a model, a pen can be a model in discribing steady-state on the axon, he will take your mp3 recorder as a model of linear association in synaptic connections, he don't bother to draw on his hand for demonstration; You can never get high scores on his exam, because it's impossible for you to fully understand a genius's thoughts...

When the old neuroscience student-fellows who graduated in 1980s, we still have the common topic of Paul Adams. What I know is that he is Fellows of the Royal Society. He is the discoverer of M-current. But then he give up his lab because he don't like the department's request for him to do something he don't like. He keeps his lectures going on all the years, to neuroscience graduate students, also to under-graduates.

Here is a interesting website of his current research, which really kind of have his style inside. "SYNAPTIC DARWINISM": http://www.syndar.org/

Shocks for neuroscientists: Doubts About Quantal Analysis

My attention was drawn when I saw this title"Long-held theory is in danger of losing its nerve:
Doubts raised over influential work on neurotransmitter release." What? Doubt about quantal release?

I went to this paper directly "Doubts About Quantal Analysis". Who dare to say this? And it's only one Author called Ninio. This will definitely be an interesting discussion. It'll be more intereting if our famous and brilliant Paul Adams is here. David came to talk to me, but he stopped just when he is about to leave, because he kind of saw the title: "What? Doubts About Quantal Analysis?" He took over my mouse and read the whole article briefly. When leaving, he said: "Paul Adams will be'd like to discuss that with you very much." "Do you believe that theory?" I asked. "Oh, the quantal release is definitely correct. But ..." I forgot the words. But what he mean is that there's no doubt about the theory, but anyone can easily come up to doubt about the data.

But you know what? Paul Adams not only knew this before us, he is even involved in the discussion!

See here:
But that rebuttal is "unconvincing, though artful", claims Paul Adams, a neurobiologist at Stony Brook University in New York. "Ninio did the best he could in view of the fact that he did not have access to the original data." Adams describes the Ninio paper as "very useful", saying that published discussions of this issue have not been as sceptical as they should have been. "The 'sheep' mentality is alive and well even at the summits of neuroscience," he says.
http://www.nature.com/nature/journal/v449/n7159/full/449124b.html

Henri Korn ( cited from a news in Oct, 1998)

Henri Korn is a French neurobiologist of international reputation known for his intellectual passion, deep knowledge of American culture and his accomplishments in both neuroscience and the humanities. For many years a visiting research professor at UB, Korn is director of the Laboratory of Cellular and Molecular Neurobiology at the Pasteur Institute in Paris.

Although his principal research is in synaptic transmission and the functional organization of the central nervous system, Korn also is a longtime serious student of philosophy and literature who in recent years has become increasingly concerned with the interplay between scientific and humanistic inquiry.

Jackson describes Korn as "a true intellectual, a man with a relentless passion for inquiry into and discussion of ideas. His knowledge of American culture is profound," Jackson said, "not just because he is a passionate reader or because of the many years he lived in the United States while doing research at UB and the Albert Einsten Medical Center. It's also because he has traveled widely and has an uncanny ability to get ordinary people to take him into their lives and let him see what they're really about."

Although his research at Pasteur focuses on the way the nerves "talk" to each other, he sees scientific inquiry not as a realm apart, but as part of the world of ideas and society, which, Jackson said, was why he was invited to give the lecture.

Korn received medical and doctoral degrees from the University of Paris. From 1991-93, he was scientific advisor to French Secretary of Defense Pierre Joxe. In 1992, he was awarded the Richard Lounsbery Prize by the National Academy of Science and the Académie des Sciences. He was elected to the Academia Europaea (1989), the French Academy of Sciences (1990) and the European Academy of Arts, Sciences and Humanities (1995).

A good lure

12 million per year for 10 years, Japan is crazy. Low-level scientists, immunologist, let's go to one of these institutions...

Kyoto University
Institute for Integrated Cell-Material Sciences
To understand and control chemical and physical processes at the cellular scale

Tohoku University
Research Center for Atom, Molecule, Materials
To promote the development of new materials, particularly bulk glass

University of Tokyo
Institute for the Physics and Mathematics of the Universe
To study basic questions about the origin, composition, and fate of the universe

Osaka University
Immunology Frontier Research Center
To merge imaging and immunology to study immune cell activity in vivo

National Institute for Materials Science
International Center for Materials Nanoarchitectonics
To study and control materials at the nano scale

News in gene regulation

"Rather than relying on mutations in a particular gene to help us digest roots and tubers better, researchers studying the evolution of starch digestion have found that the human genome simply made more copies of the gene in question."
http://www.sciencemag.org/cgi/content/full/317/5844/1483a?etoc

Reminder of Common knowledge

What is Blue/White colony selection (or screening) and how does it work?

The plasmid (or vector) used (pUC19 here) contains a gene called LacZ. The LacZ gene codes for the production of an enzyme called beta-galactosidase. This enzyme is made up of two fragments, alpha and omega. When the two fragments are associated they form a functional enzyme. Normally beta-galactosidase metabolizes galactose producing two products, lactose and glucose. Beta-galactosidase converts other substrates such as X-Gal (5-bromo-4-chloro-3-indolyl-[beta]-D-galactopyranoside) into a colored product. X-Gal is a colorless modified galactose sugar, however, when it is metabolized by beta-galactosidase the products are a bright blue.

In order for the gene to be actively transcribed from the DNA and for the enzyme to be produced, an activator called IPTG (isopropyl-[beta]-D-thiogalactopyranoside) must be added. Both X-Gal and IPTG are delivered to the bacteria through the growth medium (generally a supplemented and enriched bacto-agar blend).

Within the LacZ gene there are multiple cloning sites where the plasmid may be cut and DNA may be added. This produces a plasmid with foreign DNA located within the LacZ gene. When transcription of the gene is activated by IPTG the foreign DNA that has been inserted is transcribed as well. When the gene is later translated into the enzyme the inserted DNA is translated as well. Because of its location within the enzyme the foreign DNA's translated protein product disrupts activity and function of the enzyme. The disrupted enzyme activity is observed as a white bacterial colony. (If the enzyme is functioning fully each colony is a bright blue color.) Very small inserts of foreign DNA may lead to light blue colonies.

http://www.geocities.com/jsonnentag/iguana/bluwht.htm

The last sentence "Very small inserts of foreign DNA may lead to light blue colonies. " actully still didn't explain our phenomena. Different things.

My strange cloning story

It's just a very simple cloning: swap fragment A and B (both ~500bp), link A_up to B_dn, and B_up to A_dn. I begin making this construct with my PI since I just get into my current lab in June. To advance the rate of success, each of us did parallel experiments.

At first we just cut the two ends with KpnI and NheI, and the middle with SgrAI. Ironically each of us tried several times, but still never got the right thing. David think that maybe KpnI, which ends up a 5' overhang, is not a good enzyme for this cloning (although he it seemed fine in the past). He just want to give up and change to our 2nd stratagy: use PCR methods and change restriction sites by primers.

I insist on trying another enzyme instead of KpnI: Acc65I, which gives a 3' overhang. David don't favor trying that at all. So I did that on my own. But it still turned out to be not working. So I just moved along with him. One month already passed here.

David finished ligating the PCRed up and down fragments together, then went to vocation and gave everything to me. It's such a great day that day, I went out to hug every one in the lab, because finally I got the right A_up-B_dn thing! But strangely, none of the white colonies in the B_up-A_dn one works.

I did another set of PCR, digestion, phosphrylation, ligation (3 way including a blunt end), finally 2 out of the 18 colonies seem to work! The sequencing result is exciting but also dissappointing, it's the right swapped sequence, only with 1 PCR error! I picked another set of white colonies, again, 1 PCR error(different PCR reaction)!

I repeated the whole set of experiments again. I picked up all the possible white colonies, 2 out of the 28 got the insert, but still, PCR error again! You can't go to criticize the PCR protocol, because we already perfect it very much. We ran it only 15 cycles to turn down errors. You are just 1 um away from success, and it just seems to be a question of probability. Things are driving you crazy. I believe I am the one that luck tried to avoid.

David come back and repeated the whole thing. Funny enough, it's the same result, you got white colonies, you get 1 out of 18, and the sequencing result tells you 1 PCR error again.

Things beginning to change here. David did something that any molecular scientist can imagine. He went to pick up some blue colonies and give me to do the mini-prep and diagnostic cuts. You know what? The blue colonies got inserts!!!!

Here is our hypothesis:
1. Because our fragments are too short. It's not enough to disturb the LacZ gene to express beta-galactosidase. Thus even though it got inserts, it still form blue colony.
2. In the opposite, for those got inserts but form white colonies, it's because the frame shift in the insert totally disruptted the protein. As a result, actully all the white colonies are the ones with PCR error inside!

Something you can never believe: Blue white selection, blue is right!

Sleep, Morvan's Syndrome, and Potassium channels

http://med.ege.edu.tr/~norolbil/2001/NBD14801.html

Morvan’s syndrome (Ms) is a rare disease characterised by neuromyotonia, autonomic, central nervous system (CNS) involvement and endocrine dysfunction. Although existence of antibodies against voltage-gated potassium channels (VGKCs) has been recently established, their causative role in disease etiology has not been investigated.

Sleep and VGKCs:

Kv3.1 is a voltage-gated, fast activating/deactivating potassium channel and is expressed in fast-spiking, parvalbumin-containing interneurons in cortex, hippocampus, striatum, the thalamic reticular nucleus and in several nuclei of the brain stem. They are known to get involved in generation and maintenance of cortical fast gamma and slow delta oscillations recorded by EEG during sleep (15). This seems to be the only well-defined contribution of VGKC to sleep physiology.

Sleep and calcium activated potassium channels:

The most direct evidence of potassium channels in sleep physiology comes from apamin. Intraventricular injections of low doses of apamin, a specific blocker of a class of calcium-activated potassium channels, induce experimental insomnia, a long-lasting suppression of deep slow sleep and paradoxical sleep. Apamin especially suppresses REM sleep period (4,8). These features are quite reminiscent of the specific sleep disorder observed in Ms patients. Moreover, basal forebrain cholinergic neurons express neurotensin receptors and neurotensin blockade of calcium activated potassium channels, in this location, is a potential physiological mechanism whereby this peptide may evoke alterations in the cortical arousal, sleep-wake cycle, and theta rhythm (14). Calcium activated potassium channels of thalamic reticular nuclei neurons are also known to play role in production of spindles (a hallmark of sleep observed on EEG recordings) during sleep via thalamocortical projections (6,10). However, antibodies against this kind of potassium channel has not been reported to be detected in serum samples of Ms cases.

a suggested explanation for this situation could be as follows: Inhibition of potassium channels in the habenulointerpeduncular system may presumably result in increased acetylcholine secretion (just as it does in neuromuscular junction) and this may continously activate the interpeduncular nucleus. The extraordinary hyperactivity of this nucleus may give rise to increased activation of monoaminergic system (especially median raphe), overwhelm the cholinergic system and thus cause an increased vigilance state distinguished by stage 1 non-REM sleep characteristics. However, information about the exact role of VGKCs in this region is currently lacking and excitatory effects of acetylcholine on interpeduncular nucleus is questionable

endocrine dysfunction of Ms cases may be caused by antibodies against peripheral VGKCs rather than thalamic nuclei. It has well been established that potassium channels are major constituents of both melatonin and norepinephrine secretion mechanisms and inhibition of these channels may dramatically alter the serum levels of both hormones

Morvan's syndrome: peripheral and central nervous system and cardiac involvement with antibodies to voltage-gated potassium channels (2001 Brain)

Morvan's ‘fibrillary chorea’ or Morvan's syndrome is characterized by neuromyotonia (NMT), pain, hyperhydrosis, weight loss, severe insomnia and hallucinations. We describe a man aged 76 years with NMT, dysautonomia, cardiac arrhythmia, lack of slow-wave sleep and abnormal rapid eye movement sleep. He had raised serum antibodies to voltage-gated K⁺ channels (VGKC), oligoclonal bands in his CSF, markedly increased serum norepinephrine, increased serum cortisol and reduced levels and absent circadian rhythms of prolactin and melatonin. The neurohormonal findings and many of the clinical features were very similar to those in fatal familial insomnia, a hereditary prion disease that is associated with thalamic degenerative changes. Strikingly, however, all symptoms in our MFC patient improved with plasma exchange. The patient died unexpectedly 11 months later. At autopsy, there was a pulmonary adenocarcinoma, but brain pathology showed only a microinfarct in the hippocampus and no thalamic changes. The NMT and some of the autonomic features are likely to be directly related to the VGKC antibodies acting in the periphery. The central symptoms might also be due to the direct effects of VGKC antibodies, or perhaps of other autoantibodies still to be defined, on the limbic system with secondary effects on neurohormone levels. Alternatively, changes in secretion of neurohormones in the periphery might contribute to the central disturbance. The relationship between VGKC antibodies, neurohormonal levels, autonomic, limbic and sleep disorders requires further study.

Sleep v.s. potassium channel

Look over the topics on flies sleeping through the years: First peole found flies do sleep! Then people found the controlling location related to the mushroom body. (It's not surprising because people say the mushroom body in the fly is like the hippocampus in mammals. It's a little surprising is that an "iso-hippocampus" can have the role of controlling sleep.) Later on people found a signal pathway seem to be playing a very important role: EGFR/ERK, Rho signaling.

But what's interest me more here is, potasium channels, esp. Kv4.2 is involved!!

"What are the identities of the downstream targets for EGFR-ERG signaling in the TriC region of the fly brain? ERK directly phosphorylates the potassium channel Kv4.2 (ref. 12). Phosphorylated ERK seems to be expressed in the processes, but not in the soma of the TriC neurons. Thus, the final target of this signaling pathway may well be changes in electrical activity or synaptic transmission in these neurons. This suggestion fits nicely with work in Drosophila indicating that a mutation in the potassium channel Kv1.4 also produces abnormalities in sleep maintenance."

"Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency."

"It is possible that the mns mutation, by affecting an ion channel that controls membrane repolarization, may be close to the core cellular mechanisms of sleep. In mammals, potassium channels are involved in the generation of sleep rhythms. It is not known whether human extreme short sleepers have mutations in depolarization or voltage-dependent potassium channels. However, in Morvan's syndrome, a rare autoimmune disorder with central nervous system symptoms, marked sleeplessness has been associated with autoantibodies against voltage-dependent potassium channels that may have crossed the blood−brain barrier. The finding that a point mutation in a voltage-dependent potassium channel produces an extreme short-sleeping phenotype with preserved performance is relevant..."

Another 2 papers:

Increased motor drive and sleep loss in mice lacking Kv3-type potassium channels. (2004 Genes Brain Behav)

Sleep EEG in mice that are deficient in the potassium channel subunit K.v.3.2. ( 2002 Brain Research)

How flies sleep??!!!

Correlates of sleep and waking in Drosophila melanogaster. (2000 Science)
Shaw PJ, Cirelli C, Greenspan RJ, Tononi G. (UCSD)

Drosophila exhibits a circadian rest-activity cycle, but it is not known whether fly rest constitutes sleep or is mere inactivity. It is shown here that, like mammalian sleep, rest in Drosophila is characterized by an increased arousal threshold and is homeostatically regulated independently of the circadian clock. As in mammals, rest is abundant in young flies, is reduced in older flies, and is modulated by stimulants and hypnotics. Several molecular markers modulated by sleep and waking in mammals are modulated by rest and activity in Drosophila, including cytochrome oxidase C, the endoplasmic reticulum chaperone protein BiP, and enzymes implicated in the catabolism of monoamines. Flies lacking one such enzyme, arylalkylamine N-acetyltransferase, show increased rest after rest deprivation. These results implicate the catabolism of monoamines in the regulation of sleep and waking in the fly and suggest that Drosophila may serve as a model system for the genetic dissection of sleep.

Rest in Drosophila is a sleep-like state. （2000 Neuron）
Hendricks JC, Finn SM, Panckeri KA, Chavkin J, Williams JA, Sehgal A, Pack AI.

To facilitate the genetic study of sleep, we documented that rest behavior in Drosophila melanogaster is a sleep-like state. The animals choose a preferred location, become immobile for periods of up to 157 min at a particular time in the circadian day, and are relatively unresponsive to sensory stimuli. Rest is affected by both homeostatic and circadian influences: when rest is prevented, the flies increasingly tend to rest despite stimulation and then exhibit a rest rebound. Drugs acting on a mammalian adenosine receptor alter rest as they do sleep, suggesting conserved neural mechanisms. Finally, normal homeostatic regulation depends on the timeless but not the period central clock gene. Understanding the molecular features of Drosophila rest should shed new light on the mechanisms and function of sleep.

Reduced sleep in Drosophila Shaker mutants. (2005 Nature)
Cirelli C, Bushey D, Hill S, Huber R, Kreber R, Ganetzky B, Tononi G. (U Wisconsin Madison)

Most of us sleep 7-8 h per night, and if we are deprived of sleep our performance suffers greatly; however, a few do well with just 3-4 h of sleep-a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, we performed mutagenesis in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, we found minisleep (mns), a line that sleeps for one-third of the wild-type amount. We show that mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. We then show that mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, we show that short-sleeping Shaker flies have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency.

Dopaminergic modulation of arousal in Drosophila. (2005 Curr Biol)
Andretic R, van Swinderen B, Greenspan RJ. (UCSD)

Changes in dopamine levels differentially affect arousal for behaviors of varying complexity. Complex behaviors, such as visual perception, degenerate when dopamine levels are either too high or too low, in accordance with the inverted-U hypothesis of dopamine action in the mammalian brain. Simpler behaviors, such as sleep and locomotion, show graded responses that follow changes in dopamine level.

A dynamic role for the mushroom bodies in promoting sleep in Drosophila. (2006 Nature)
Pitman JL, McGill JJ, Keegan KP, Allada R. (Northwestern)

The fruitfly, Drosophila melanogaster, exhibits many of the cardinal features of sleep, yet little is known about the neural circuits governing its sleep. Here we have performed a screen of GAL4 lines expressing a temperature-sensitive synaptic blocker shibire(ts1) (ref. 2) in a range of discrete neural circuits, and assayed the amount of sleep at different temperatures. We identified three short-sleep lines at the restrictive temperature with shared expression in the mushroom bodies, a neural locus central to learning and memory. Chemical ablation of the mushroom bodies also resulted in reduced sleep. These studies highlight a central role for the mushroom bodies in sleep regulation.

Sleep in Drosophila is regulated by adult mushroom bodies. (2006 Nature)
Joiner WJ, Crocker A, White BH, Sehgal A. (Upenn Medical)

Sleep is one of the few major whole-organ phenomena for which no function and no underlying mechanism have been conclusively demonstrated. Sleep could result from global changes in the brain during wakefulness or it could be regulated by specific loci that recruit the rest of the brain into the electrical and metabolic states characteristic of sleep. Here we address this issue by exploiting the genetic tractability of the fruitfly, Drosophila melanogaster, which exhibits the hallmarks of vertebrate sleep. We show that large changes in sleep are achieved by spatial and temporal enhancement of cyclic-AMP-dependent protein kinase (PKA) activity specifically in the adult mushroom bodies of Drosophila. Other manipulations of the mushroom bodies, such as electrical silencing, increasing excitation or ablation, also alter sleep. These results link sleep regulation to an anatomical locus known to be involved in learning and memory.

Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila （2007 Nature）
Krisztina Foltenyi1,Ralph J Greenspan & John W Newport

Epidermal growth factor receptor (EGFR) signaling in the mammalian hypothalamus is important in the circadian regulation of activity. We have examined the role of this pathway in the regulation of sleep in Drosophila melanogaster. Our results demonstrate that rhomboid (Rho)- and Star-mediated activation of EGFR and ERK signaling increases sleep in a dose-dependent manner, and that blockade of rhomboid (rho) expression in the nervous system decreases sleep. The requirement of rho for sleep localized to the pars intercerebralis, a part of the fly brain that is developmentally and functionally analogous to the hypothalamus in vertebrates. These results suggest that sleep and its regulation by EGFR signaling may be ancestral to insects and mammals.

Soporific signaling: how flies sleep through the night. （2007 Nature neurocience）
Colwell CS

A new study identifies a growth factor signaling pathway involved in sleep regulation and consolidation in this model. Inhibiting this pathway causes a sleep pattern that is similar to insomnia in humans.

My "publications" --- a link to my unusual friend

http://jwu-ht.blogspot.com/2007/09/erpdb-database-of-erp-data-for.html

Introduction about Qinghong Yan

I'm currently a PHD student in SUNYsb Neuroscience graduate program. My belief is science. My current idealistic aim is to be a knowledgeable scientist. My current realistic aim is to successfully complete my PHD study in 5 years (that is, before my 26th birthday) and get some good publications.

This blog is to organize the knowledge related to my research for myself, also by the way, to share with others, if there're something in common between us.

I might also would like to publish some stupid comments myself.